(a) Field of the Invention
The present invention relates to a group of antitumor agents which by themselves are not toxic and exhibit an improved tumor selective cytotoxic action due to their preferential accumulation in tumor tissue only after subsequent administration of nontoxic substrate components, and a method for treating cancer by administration of these antitumor agents.
More specifically, it relates to the use of non-toxic antitumor agents consisting oxidoreductases, such as xanthine oxidase, which are chemically conjugated with polymers, and time lapse injection of the enyme's substrates, such as hypoxanthine or xanthine.
(b) Description of the Prior Art
Several antitumor agents such as mitomycin and doxorubicin have been found to exhibit their antitumor effect based on their capability of generating reactive oxygen molecular species. An earlier work by R. Bray and his co-workers (Nature, vol. 182, p. 1144-1146, 1958) and more recently T. Yoshikawa and his co-workers (Cancer Res., vol. 55: p. 1617-1620, 1995) reported that the antitumor activity of xanthine oxidase (hereinafter referred to as “XO”) is achieved probably via the generation of reactive oxygen molecular species. However, a more critical reevaluation of the antitumor effect of native XO by R. Bray and J. C. Swann showed that the effect was insignificant (Structure and Bonding, vol. 11, p. 107-144, 1972, published by Elsevior, a note added in footnote of page 112). This was also confirmed again by the present inventors.
Reactive oxygen molecular species generated from those antitumor drugs exhibit an antitumor effect based on their highly cytotoxic nature. However systemic distribution of those drugs causes undesirable side effects (J. Clin. Invest., vol. 98, p. 1253-1260, 1996). For instance, native XO readily binds to blood vessels after the administration into blood due to its high binding affinity to vascular endothelial cells (Biochem. J., vol. 289, 523-527, 1993). The binding of XO to blood vessels is expected to cause serious side effects such as: i) The superoxide anion radical generated from XO would oxidatively damage blood vessels; ii) The reaction between the superoxide and endogenously formed nitric oxide leads to dilatation of the blood vessels and lowers the blood pressure or thus regulates the blood pressure (Pharmacol. Rev., vol. 43, p. 109-142, 1991), which would cause hypertension due to lowered level of nitric oxide in the blood vessels (Proc. Natl. Acad. Sci. USA, vol. 88, p. 10045-10048, 1991), iii) The reaction product of superoxide and nitric oxide, namely the peroxynitrite (ONOO—) further oxidatively damages the blood vessels. Therefore it is not advisable to employ native XO for clinical use. In addition, endogenous anti-XO antibody (Brit. J. Biomed. Sci., vol. 51, 124-127, 1994) may reduce the activity of XO after intravenous injection.
To enhance the drug efficacy while reducing the systemic side effects, it is necessary to deliver this antitumor enzyme selectively to the tumor tissue. The inventors previously found that macromolecular drugs and lipids preferentially accumulate in the tumor tissue compared with other normal organs, and furthermore they are retained in the tumor tissue for a longer period. This phenomenon is called the EPR effect (enhanced permeability and retention effect), Cancer Res., vol. 46, p. 638-792, (1986). The enhanced therapeutic efficacy and the reduction of side effects could be achieved by increasing the molecular weight of the antitumor agent (J. Controlled Rel., vol 19, p. 315-324, 1992).
The object of the present invention is to provide a group of antitumor agents which exhibit an improved tumor selective accumulation and therefore an improved tumor selective cytotoxicity.
This object is met by the present invention according to which an antitumor effect is generated by a combination of an oxidoreductase, which is chemically conjugated with biocompatible polymer, and a substrate for the oxidoreductase.
The antitumor agent according to the present invention is a combination of an active enzyme component (A) and of its substrate (B). The active enzyme component (A) is an oxidoreductase, which is chemically conjugated with a polymer. Upon administration of (A) and later on administration of (B), an active molecular species (C), such as a peroxide, is formed.
The active enzyme component (A), by its polymer conjugation, possesses a tumor targeting property. Namely, the antitumor agent exhibits a selective accumulation in tumor tissue and exerts an antitumor action if a known substrate (B) for the active enzyme component (A) is injected thereafter. Due to the enzyme reaction, an active free radical components (C) (O2.— and H2O2 are formed. Both xanthine oxidase conjugated with poly(ethylene glycol) (A) and its substrate (B) show no toxicity by themselves. The potent antitumor activity is only apparent when substrate (B) is separately administered later. By doing so, less systemic toxicity is seen while exhibiting a remarkable antitumor activity. Thus the present invention offers great benefit.